CCK Gene Research Articles

Cholecystokinin (CCK) Gene as a Possible Risk Factor for Smoking: A Replication in Two Independent Samples

Background. CCK is a satiety neuropeptide. Animal studies have shown that both acute and chronic exposure to nicotine results in weight loss which is associated with an increase in hypothalamic CCK and that CCK antagonists ameliorate symptoms of nicotine withdrawal. A major detriment to smoking cessation, especially in women, is the fear of gaining weight. These observations suggested that genetic variants in the CCK gene might be a possible risk factor for smoking.Methods. To test this hypothesis we examined the association of the C-45T promoter polymorphism in the Sp1 binding region of the CCK gene with smoking and BMI in two independent groups of subjects.Results. Group 1 consisted of 191 Caucasian women participating in an obesity study. The T allele was present in 15% of women who had never smoked, 20% of ex-smokers, and 58% of current smokers, P ≤ 0.0014. The T allele was present in 26.8% of ever-smokers (ex-smokers + current smokers). There was no association with BMI. Group 2 consisted of 725 parents of twins from the Minnesota Twin and Family Study of substance abuse. Logistic regression analysis showed that a diagnosis of nicotine dependence was significantly associated with the T allele (P ≤ 0.002) and with gender (males > females) (P ≤ 0.001), but not with BMI (P ≤ 0.68). The T allele was present in 15.9% of parents who had never smoked and 24.7% of ever-smokers, very similar to the results for group 1.Interpretation. These results are consistent with a role of the CCK gene as a risk factor for smoking.

Polymorphisms of the CCK, CCKAR and CCKBR genes: an association with alcoholism study.

Cholecystokinin (CCK) plays an important role in the functioning of the central nervous system via an interaction with dopamine and other neurotransmitters. The dopaminergic system has been implicated in the maladaptive behaviors associated with drugs of abuse (e.g., alcohol and cocaine). We analyzed genetic variations in the promoter and coding regions of the CCK, CCKA-receptor (CCKAR) and CCKB-receptor (CCKBR) genes, and performed association analyses with alcoholism. A total of 214 Japanese male patients with alcoholism (93 with delirium tremens, 49 with hallucinations, 38 with seizures and 83 without any of these symptoms) and 98 age-matched Japanese male controls were examined using PCR-based Single Strand Conformational Polymorphism (SSCP) analysis. A total of 8 variants in the CCK gene, 11 variants in the CCKAR gene and 9 variants in the CCKBR gene were detected in the present study. Nominally significant differences between alcoholics and controls were found at the -85 locus of the CCKAR gene (p = .035). In addition, patients displaying hallucinations showed a higher frequency of the homozygous genotype of the allele (GT)8 at the -388 locus of the CCKAR gene (p = .042) and homo- and heterozygous genotypes of the T allele at the -333 locus (p = .025), relative to other patients. However, these differences were not significant after Bonferroni correction. Our data suggest that polymorphisms of the CCK, CCKAR and CCKBR genes do not play a major role in alcohol withdrawal symptoms (even though significant associations were found among polymorphisms at the -388 and -333 loci of the CCKAR gene and hallucinations, the rate was nonsignificant after Bonferroni correction).

Peptones Stimulate Intestinal Cholecystokinin Gene Transcription via Cyclic Adenosine Monophosphate Response Element-Binding Factors

Cholecystokinin (CCK) is a potent intestinal hormone that regulates several digestive functions. Despite the physiological importance of CCK, the cellular and molecular mechanisms that govern its synthesis and secretion are not completely identified. Peptones, which are fair counterparts of the protein fraction in the intestinal lumen, are good stimulants of CCK secretion. We have previously shown that peptones activate CCK gene transcription in STC-1 enteroendocrine cells. The DNA element(s) necessary to induce the transcriptional stimulation was preliminary, localized in the first 800 bp of the CCK gene promoter. In the present study, we identify a DNA element [peptone-response element (PepRE)] essential to confer peptone-responsiveness to the CCK promoter, and we characterize the transcription factors implicated. Localization of the PepRE between -93 and -70 bp of the promoter was established using serial 5'-3'deletions. Systematic site-directed mutagenesis demonstrated that the core PepRE sequence, spanning from nucleotide -72 to -83, overlapped with the putative AP-1/CRE site. Mutations in the core sequence dramatically decreased peptone-responsiveness of CCK promoter fragments. The PepRE functioned as a low-affinity CRE consensus site, binding only transcription factors of the CREB family. Overexpression, in STC-1 cells, of a dominant-negative protein (A-CREB), that prevented the binding of CREB factors to DNA, completely abolished the peptone-induced transcriptional stimulation. Peptone treatment did not modify the nature and the abundance of proteins bound to the PepRE but led to increased phosphorylation of the CREB factors. In conclusion, the present study first demonstrates that CCK gene expression is under the control of protein-derived nutrients in the STC-1 enteroendocrine cell line.

Peptones Stimulate Intestinal Cholecystokinin Gene Transcription via Cyclic Adenosine Monophosphate Response Element-Binding Factors

Cholecystokinin (CCK) is a potent intestinal hormone that regulates several digestive functions. Despite the physiological importance of CCK, the cellular and molecular mechanisms that govern its synthesis and secretion are not completely identified. Peptones, which are fair counterparts of the protein fraction in the intestinal lumen, are good stimulants of CCK secretion. We have previously shown that peptones activate CCK gene transcription in STC-1 enteroendocrine cells. The DNA element(s) necessary to induce the transcriptional stimulation was preliminary, localized in the first 800 bp of the CCK gene promoter. In the present study, we identify a DNA element [peptone-response element (PepRE)] essential to confer peptone-responsiveness to the CCK promoter, and we characterize the transcription factors implicated. Localization of the PepRE between −93 and −70 bp of the promoter was established using serial 5′-3′deletions. Systematic site-directed mutagenesis demonstrated that the core PepRE sequence, spanning from nucleotide −72 to −83, overlapped with the putative AP-1/CRE site. Mutations in the core sequence dramatically decreased peptone-responsiveness of CCK promoter fragments. The PepRE functioned as a low-affinity CRE consensus site, binding only transcription factors of the CREB family. Overexpression, in STC-1 cells, of a dominant-negative protein (A-CREB), that prevented the binding of CREB factors to DNA, completely abolished the peptone-induced transcriptional stimulation. Peptone treatment did not modify the nature and the abundance of proteins bound to the PepRE but led to increased phosphorylation of the CREB factors. In conclusion, the present study first demonstrates that CCK gene expression is under the control of protein-derived nutrients in the STC-1 enteroendocrine cell line.

Regulation of neuronal cholecystokinin gene transcription

Cholecystokinin (CCK) is a neuroendocrine peptide expressed in I-cells of the small intestine and in central and peripheral neurons. Whereas intestinal CCK is involved in the release of pancreatic enzymes and the contraction of the gallbladder, cerebral CCK is implicated in a variety of functions, such as feeding behaviour, anxiety and memory. The expression of CCK is developmentally regulated. Brain CCK mRNA levels are low before birth, but increase markedly shortly after birth and reach adult like patterns of expression three weeks after birth during the final maturation of the central nervous system. In the adult, several substances induce neuronal CCK mRNA expression via activation of transcription factors binding to regulatory elements in the CCK promoter. Recent studies have examined the signaling pathways, transcription factors and regulatory elements involved in cAMP, fibroblast growth factor-2, and calcium-induced CCK gene transcription in neuronal cells. The review describes the signaling pathways and transcription factors involved in neuronal CCK gene transcription.

Control of CCK gene transcription by PACAP in STC-1 cells.

The mechanisms by which neuroendocrine stimulants regulate CCK gene transcription are unclear. We examined promoter activation by pituitary adenylate cyclase-activating polypeptide (PACAP), a known CCK secretagogue, in the enteroendocrine cell line STC-1. The promoter region from -70 to -87 bp, relative to the transcriptional start site, contains a composite calcium/cyclic AMP response element (CRE)/activator protein 1 (AP1) site that may bind CRE binding protein (CREB) and AP1. PACAP (with IBMX) stimulated expression of an 87-bp construct 3.35+/-0.36-fold but had no effect on a -70 construct. The effect was blocked by the protein kinase A inhibitor H-89 and by a dominant-negative CREB plasmid. Mutation of the CRE/AP1 site to a canonical CRE site did not affect the response to PACAP, but mutation to a canonical AP1 site prevented it. CREB phosphorylation was increased after PACAP treatment. Electrophoretic mobility shift assay and supershift analysis revealed that CREB and not AP1 bound to the CRE/AP1 site and that PACAP increased the proportion of phosphorylated CREB that was bound. We conclude that PACAP increases CCK gene expression via a cAMP-mediated pathway involving CREB phosphorylation by protein kinase A and activation of a composite CRE/AP1 site.

Function of the C-36 to T polymorphism in the human cholecystokinin gene promoter.

Cholecystokinin (CCK) is the most abundant neuropeptide in the mammalian brain, and in man significant quantities are expressed in all regions of the brain.1,2 Therefore, CCK has been implicated in a variety of CNS functions-such as feeding behavior, anxiety, analgesia and memory functions as well as psychiatric disease like panic disorder and schizophrenia (for review, see2,3). Recently, a number of studies have indicated that a C-36 to T transition in the CCK gene promoter Sp1 element4 (Figure 1) is associated with alcoholism and withdrawal symptoms as well as panic disorder.5-7 Moreover, it has been proposed that the polymorphism plays a direct role in the pathogenesis of the disorders by decreasing the expression and synthesis of CCK peptides. The significance of these findings is still unclear and other studies have failed to demonstrate linkage between the polymorphism and alcoholism.8 In this study we examined the function of the C-36 to T transition in transcription of the human CCK gene. We demonstrate that substitution of the C-36 residue causes a slight reduction of Sp1 and Sp3 binding, but this has no effect on transcription in vivo. Moreover, no difference in the response to physiological stimuli was observed. Taken together the results show that the C to T polymorphism does not play a direct role in the pathogenesis of either alcoholism or panic disorder and that a putative association to these disorders is likely to be the result of co-segregation with a linked mutation.

72. A genetic variant of CCK promoter region may link to negative symptom of schizophrenia

The finding that neuropeptide cholecystokinin (CCK) co-localized with dopamine in central nervous system has made CCK an important candidate gene for schizophrenia research. Several previous studies have found reduced CCK level in cerebrospinal fluid of schizophrenia patients. A more recent study reported a marked decrease of CCK mRNA in the frontal and temporal cortex of schizophrenia. In present study, mutation screens were carried out on the CCK gene by means of single strand conformational polymorphism (SSCP). A C to T substitution was found in transcription factor Sp1 binding site of the CCK promoter region. Such variation can affect the gene expression by altering the transcription factor binding. The T allele variation was detected in 24% of the 85 DSM-III-R or DSM-IV schizophrenics, and 16% of 247 geographically matched unscreened controls. While the higher frequency of T alleles are found in schizophrenia subjects, but the association between them was not statistically significant compared with controls (χ2 = 2.30, p > 0.1). We further analyse the schizophrenia subjects by dividing them into subgroups according to the score on the Scale for Assessment of Positive Symptoms (SAPS 0–5, SAPS 6–10) and Scale for Assessment of Negative symptoms (SANS 0–10, SANS 11–20). The results demonstrated a statistically significant association between T allele and schizophrenia patients with SANS score greater than 11 (χ2 = 4.13, p < 0.05). The finding indicate that the C to T substitution in the transcription factor Sp1 binding site of CCK promoter region may related to the negative symptoms of schizophrenia.

Genetic Polymorphism of the CCK Gene in Patients With Alcohol Withdrawal Symptoms

Cholecystokinin (CCK) is considered to play an important role in the central nervous system via its interaction with other neurotransmitters such as dopamine, serotonin, gamma-aminobutyric acid, substance P, and enkephalins. We investigated the relationship between the C to T substitution in the Sp1 binding cis-element of the CCK gene promoter region (at position -45 numbered from initiation codon) and alcohol withdrawal symptoms. We examined 214 Japanese men with alcoholism (93 with delirium tremens, 49 with hallucination, 38 with seizure, and 93 with none of these symptoms) and 98 age-matched Japanese male controls by using a polymerase chain reaction-based single strand conformational polymorphism analysis. Patients who displayed hallucination were significantly more likely to possess the C allele than control subjects (chi2 = 8.17, p = 0.017, Bonferroni correction: p = 0.064). In addition, we investigated the influence of CCK gene polymorphism on alcohol consumption among the control subjects but found no significant relationship. Our data suggested that the C allele at -45 locus of the CCK gene was higher in patients with hallucination than the control group at a rate that was not quite significant after Bonferroni correction for multiple testing.

Association between polymorphism of the cholecystokinin gene and idiopathic Parkinson's disease.

Parkinson's disease (PD) is characterized by major alterations of neurotransmitter activity due to damage of the substantia nigra. Changes in neuropeptide concentration within the basal ganglia may play an important role in the putative dopaminergic-peptidergic interactions associated with the disease. Cholecystokinin (CCK) modulates the release of dopamine in the mesolimbic pathway and affects dopamine-related behavior. We analyzed genetic variations in the CCK gene, in both the coding and promoter region, in order to investigate the role of polymorphism in idiopathic PD. Four polymorphic sites of the CCK gene (-196G/A, -45C/T, 1270C/G, 6662C/T) were found in PD patients and controls. Complete linkage disequilibrium was observed between the -45 locus and the 1270 locus, and also a possible linkage disequilibrium was found between the -45 and -196 loci. A significant difference was found in the distributions of three identified genotypes at the -45 locus between 116 PD patients and 95 age-matched control subjects (chi2 = 7.95, p = 0.018, Bonferroni correction; p = 0.054). In addition, a significant difference was obtained amongst the three genotypic groups at the -45 locus when compared between PD patients who experienced hallucinations (n = 23) and those (n = 93) who did not (chi2 = 8.08, p = 0.018, Bonferroni correction, p = 0.126). Our data suggested that mutations at the -45 locus in the promoter region of the CCK gene may influence vulnerability to hallucinations in PD patients treated with L-dopa.

Murine prenatal expression of cholecystokinin in neural crest, enteric neurons, and enteroendocrine cells.

Cholecystokinin (CCK) is a regulatory peptide that is primarily expressed in two adult cell types: endocrine cells of the intestine and neurons of the central nervous system. To determine the ontogeny of CCK expression during intestinal organogenesis, we created a mouse strain in which the CCK gene was replaced by a lacZ reporter cassette using homologous recombination in embryonic stem cells. Initially, CCK expression in the developing intestine was limited to the myenteric plexus of the enteric nervous system. This expression pattern was widespread, extending from the proximal stomach into the colon, yet transient, being detected soon after gut tube closure [embryonic day 10.5 (E10.5)] through E15.5. Since enteric neurons are derived from the neural crest, we examined earlier (E8.5-9.5) embryos and concluded that lacZ was expressed in subpopulations of neural tube and neural crest cells. Endocrine cell expression in the intestinal epithelium occurred later, beginning at E15.5 as enteric neuronal expression was dwindling. This expression persisted to yield the adult pattern of scattered single endocrine cells in the upper small intestine. The data show that CCK is a very early marker of both neuronal and endocrine cell lineages in the developing gastrointestinal tract. Furthermore, reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that CCK receptor transcripts were detected in embryos as early as E10.5, suggesting that CCK signaling is established early in mouse development. Dev Dyn 1999;216:190-200.

Negative cooperativity between juxtaposed E-box and cAMP/TPA responsive elements in the cholecystokinin gene promoter

The promoter of the cholecystokinin (CCK) gene possesses evolutionary conserved juxtaposed E-box and cAMP/TPA responsive elements (CRE/TRE). We have examined the functional interaction of these two sites. As previously noted, c-Jun/c-Fos heterodimers greatly increase promoter activity through association with the CRE/TRE. Mutation of the E-box enhanced the activation by c-Jun/c-Fos, as well as stimulation by forskolin and bFGF, that acts through the CRE/TRE site. Moreover, c-Jun/c-Fos stimulation was inhibited by co-expression of c-Myc and Max. The results indicate that factors associating with the E-box exhibit a negative cooperative effect on the activation via the CRE/TRE element. We propose that this mechanism plays a significant role in CCK gene transcription and other genes with juxtaposed E-box and CRE/TRE.

Mitogen-activated protein kinase and protein kinase A signaling pathways stimulate cholecystokinin transcription via activation of cyclic adenosine 3',5'-monophosphate response element-binding protein.

Cholecystokinin (CCK) is a potent neuropeptide expressed in the small intestine and in the central nervous system. We have examined the effect of basic fibroblast factor (bFGF) and forskolin on CCK gene transcription and depicted the signaling pathways that lead to promoter activation. bFGF and forskolin stimulated promoter activity via a cAMP response element (CRE)/12-O-tetradecanoylphorbol-13-acetate response element (TRE) located 80 bp upstream from the transcription initiation site. In nuclear extracts from unstimulated as well as stimulated cells, only CRE-binding protein (CREB) and activating transcription factor-1 (ATF-1) bound to the CRE/TRE, and activation was associated with phosphorylation of CREB serine-133 and ATF-1 serine-63. In murine F9 cells, CREB stimulated promoter activity 10-fold in the presence of protein kinase A (PKA), and in SK-N-MC cells activation was inhibited 60-70% by a dominant negative CREB mutant. In contrast, ATF-1 had no effect in F9 cells and exhibited a dominant negative effect in SK-N-MC cells. bFGF stimulation led to phosphorylation of the p38 mitogen-activated protein kinase (MAPK), and the extracellular signal-regulated kinase (ERK) MAPK and promoter activation, phosphorylation of CREB, and GAL4-CREB-dependent transcription were selectively prevented by a dominant negative Ras-mutant, the p38 MAPK-specific inhibitor SB203580, and the MAP/ERK kinase 1 (MEK1) inhibitor PD098059. Forskolin stimulation proceeded via the PKA pathway, and to a minor extent via the p38 and ERK MAPK pathways. We conclude that bFGF and forskolin stimulate the CCK gene promoter via the CRE/TRE(-80) in the proximal promoter region. Signaling proceeds through the p38 MAPK, the ERK MAPK, and the PKA-signaling pathways, which leads to cumulative phosphorylation and activation of CREB. We propose that bFGF in combination with neurotransmitters/neuropeptides coupling to the PKA-signaling pathway play an important role in the control of CCK gene expression.

No association between C-45T polymorphism in the Sp1 binding site of the promoter region of the cholecystokinin gene and alcoholism

The activity of dopamine-containing neurons in the ventral tegmental area and nucleus accumbens may play a role in alcoholism. Cholecystokinin (CCK) co-exists in a large proportion of A10 dopamine neurons to exert some effect on dopamine-induced behavior. Recently, a C-45T polymorphism was discovered in the Sp1 binding site in the CCK gene promoter region. We investigated an association between the polymorphism and alcoholism in 209 Japanese DSM-III-R alcoholics and 113 Japanese control subjects. The patients and the control subjects had similar allele and genotype frequencies: the T allele frequencies were 0.27 in the patients and 0.28 in the control subjects and the CC, CT, and the TT genotype frequencies 0.53, 0.39, and 0.08 in the alcoholics and 0.53, 0.37, and 0.10 in the control subjects. Frequencies of clinical characteristics of Feighner's criteria for the lifetime diagnosis of alcoholism were not significantly different among the patient groups divided by the genotype. These findings indicate that the polymorphism has no major effect on the etiology of alcoholism.

Mapping of cholecystokinin transcription in transgenic mouse brain using Escherichia coli beta-galactosidase reporter gene.

Cholecystokinin (CCK), a neuro-gut peptide, occurs not only in the nervous but also in the digestive system. As a first step in elucidating whether CCK gene expression and its physiological functions co-operate in these separate organs, transgenic mice were produced using CCK promoter that directs bacterial beta-galactosidase as a reporter gene. A new transgenic vector was constructed, inserting the SV40 poly A signal 5' to the CCK promoter to impede any transcription upstream of the transgene. A 2.4 kb.p. region upstream to the transcription start site of the mouse CCK gene was used as the promoter. Transgene expression was detected first at embryonic 13.5 days in the central nervous system and increased after birth. The distribution of cells expressing beta-galactosidase transgene agreed fairly well with that of in situ hybridization. In addition, the upregulation of CCK gene expression was clearly demonstrated by expressing beta-galactosidase after injury to the brain. These results indicated that the 2.4 kb.p. of the CCK gene promoter region was sufficient to direct appropriate tissue-specific gene expression in mice.

An association of NAG levels and a mutation of the CCK gene in panic disorder patients

Levels of the enzyme N-acetyl-β-glucosaminidase (NAG) and a mutation of cholecystokinin (CCK) gene appear to be independently associated with panic disorder. We explored whether there was an association of NAG levels and a CCK mutation identified in a group of panic disorder patients. NAG was measured in 12 panic disorder patients who had a mutation of the CCK gene and 17 who did not. Urine for NAG was collected at baseline and after 3 and 6 weeks of treatment. NAG levels were lower at all three times in the patients that did not have the CCK mutation. The difference between the two groups was significant at week 6 ( P<0.02), and showed a trend toward a difference at baseline ( P<0.15).

Possible association of a cholecystokinin promotor polymorphism (CCK-36CT) with panic disorder.

We searched for mutations in the CCK gene in panic disorder with single-strand conformational polymorphism (SSCP) analysis of the three exons and promotor region of the gene. We found a C-->T transition at position -36 (CCK(-36C-->T)) in a GC box, a binding site for transcription factor Sp1, in the promotor region. The allele frequency was 0.168 (95% CI, 0.116-0.221) in 98 persons with panic disorder and 0.083 (95% CI, 0.059-0.107) in 247 geographically matched, unscreened controls. A transmission disequilibrium test based on panic disorder as the affected phenotype was nonsignificant (chi2 = 0.93), but when panic disorder or attacks were considered as affected, statistically significant transmission disequilibrium was detected (chi2 = 4.00, P < 0.05). Linkage analysis was uninformative. In exploratory analyses to search for clinical correlations, the "T" allele was found in 59% of 22 persons with panic attacks but not panic disorder, compared with 31% of those who met the criteria for panic disorder. An association between the CCK polymorphism and panic disorder cannot be considered established due to the inconsistencies in the results noted above, but if the provisional association can be replicated, the findings are consistent with CCK(-36C-->T) being a disease-susceptibility allele that alone is neither necessary nor sufficient to cause panic disorder but that increases vulnerability by acting epistatically.

Linked polymorphisms upstream of exons 1 and 2 of the human cholecystokinin gene are not associated with schizophrenia or bipolar disorder.

The evidence for a significant genetic contribution to the functional psychoses (schizophrenia and bipolar disorder) is now well established. However, in both cases, the non-mendelian mode of inheritance has made the identification of susceptibility loci particularly challenging. The neuropeptide cholecystokinin (CCK) is present both in the gut and the CNS. Studies of CCK-like immunoreactivity and CCK mRNA levels in human brains have revealed high concentrations in numerous loci and shown colocalisation of CCK with, for example, dopamine and tyrosine hydroxylase. Furthermore, antagonists of CCK-B receptors, which are found most frequently in the brain, inhibit the activity of brain dopamine neurons. Such findings suggest that, with respect to neuropsychiatric disorders, CCK is a suitable candidate for analysis using methods to detect gene variations which have the potential to affect protein structure or expression. In the present study, mutation analyses were carried out on the human CCK gene. Linked polymorphisms were found in the promoter region and in intron 1 close to the 3' mRNA splice acceptor site. However, the allele frequencies of these polymorphisms in samples of individuals affected with either schizophrenia (n=117) or bipolar disorder (n=124) did not differ from those of control subjects (n=234), suggesting that these variations do not confer a predisposition to either of the functional psychoses.

Development of an in vivo method to study bovine intestinal response to dietary manipulation at the molecular level.

Our objective was to develop an in vivo method for analysis of nutrient-gene interactions in dairy cows at the molecular level. A series of techniques was applied to study the molecular response of cells in bovine small intestine to dietary manipulation. Three Holstein cows fitted with ruminal and duodenal cannulae were used for the investigation. As part of the validation of the procedure, lipid flow to the small intestine was manipulated using an abomasal infusion technique to study the expression of the gene encoding for the gastrointestinal hormone cholecystokinin (CCK). Biopsies (10 to 25 mg) were collected from the duodenum approximately 40 to 50 cm distal from the pyloric sphincter using a fiberoptic endoscope and biopsy forceps. Total RNA was isolated from the intestinal biopsies with yields of 20 to 50 microg. Cholecystokinin gene expression was analyzed in intestinal mucosa obtained from Holstein cows after slaughter either at a local abattoir or at our research facilities and compared with CCK gene expression in the biopsy samples collected from the cannulated animals. Using the technique of northern hybridization, we found that 15 microg of total RNA was sufficient to detect a strong CCK mRNA signal in the biopsy tissue samples, whereas 30 microg of total RNA showed a slightly weaker CCK mRNA signal in the postmortem duodenal tissues collected at our research facility. In contrast, the CCK mRNA signal in postmortem duodenal tissues obtained from cows slaughtered at the local abattoir was only barely detectable with 40 microg of total RNA. We conclude that the biopsy procedure is relatively noninvasive and is useful for monitoring intestinal responses to dietary manipulation and to physiological changes, such as those that occur during lactation and with metabolic disorders. The application of the method will provide insight into nutrient-gene interactions that occur in bovine intestinal mucosa.

Evidence for the existence of CCK-producing cells in rat pancreatic islets.

Although the existence of cholecystokinin-like immunoreactivity (CCK-LI) in rat pancreas had been reported previously, it was never clearly demonstrated whether CCK is produced in rat pancreatic islets. The purpose of this study was to elucidate the source of the CCK-LI, the molecular properties of CCK, and the expression of the CCK gene in islet cells. Immunohistochemical studies of rat pancreas were carried out with different rabbit antisera against CCK-8 and CCK-related peptide including N-terminal CCK-33 (1-22) and gastrin-17, and colocalization with known islet hormones including insulin, glucagon, somatostatin, and pancreatic polypeptide was investigated. The major molecular form of CCK in the islets was determined by HPLC. RT-PCR and in situ hybridization were performed to demonstrate the presence of the CCK transcript in the pancreas. CCK-LI was found in the center of the islets, colocalized with insulin in B cells. The major molecular form of CCK in the islets was CCK-8. A 350-nucleotide fragment of PCR-amplified CCK cDNA was detected in the islet as well as the duodenum by RT-PCR. In situ hybridization showed that CCK messenger RNA was located in a large portion of the islets, and this was consistent with the immunohistochemical findings. CCK messenger RNA and immunoreactivity are expressed in adult rat pancreatic islets, indicating that CCK-producing cells are present in adult rat islets.